Cloning of an aroF allele encoding a tyrosine-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

In Escherichia coli, genes aroF+, aroG+, and aroH+ encode isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. A single base pair change in aroF causes a Pro-148-to-Leu-148 substitution and results in a tyrosine-insensitive enzyme.     

Cloning, sequence, and expression of the Drosophila cAMP-dependent protein kinase catalytic subunit gene

Genomic DNA containing the protein coding region for Drosophila cAMP-dependent protein kinase catalytic subunit has been cloned and sequenced. The probe used to detect and isolate the gene fragment was constructed from two partially complementary synthetic oligonucleotides and contains 60 base pairs that encode (using Drosophila codon preferences) amino acids 195-214 of the beef heart catalytic subunit. In reduced stringency hybridization conditions, the probe recognizes two target sites in fly genomic DNA with 85% homology. One of these sites is in the cAMP-dependent protein kinase catalytic subunit gene, which was isolated as a 3959-base pair HindIII fragment. This fragment contains all of the protein coding portion, 900 base pairs upstream of the initiator ATG, and 2000 base pairs downstream of the termination codon (TAG). The coding portion of the gene contains no introns and yields a protein of 352 amino acids. There is a 2-amino acid insertion near the N terminus of the fly protein relative to the beef and mouse enzymes. Of the remaining 350 amino acids, 273 are invariant in the three species. A probe derived from the coding sequence of the HindIII clone hybridizes strongly to a 5100-base poly(A)+ RNA and weakly to 4100- and 3400-base poly(A)+ RNAs expressed in adult flies. A 2100-base pair EcoRI genomic fragment containing the second site recognized by the 60-base pair probe has also been cloned. DNA sequence analysis demonstrates that this fragment is part of the cGMP-dependent protein kinase gene or a close homolog. The catalytic subunit gene and the cGMP-dependent protein kinase gene have been located in regions 30C and 21D, respectively, of chromosome 2.     

In vitro methylation of the 5'-flanking regions of the mouse beta-globin gene

The enzymatic methylation of the 5'-flanking region of the mouse beta-globin (major) gene containing putative regulatory regions has been investigated. In vitro methylation of this 368-base pair regulatory DNA by a DNA methyltransferase obtained from mouse erythroleukemia cells yields an asymmetric methylation pattern. Of the 10 available CG pairs, only 5-6 are modified, leading to one hemimethylated site and two apparently fully methylated sites. Only CG pairs which are localized in a 29-base pair cluster are methylated. The data suggest that a CG cluster approximately 100 base pairs upstream from the CAP site may be the in vivo site of methylation in the 5'-regulator region of the mouse beta-globin gene.     

Characterization of the DNA binding region recognized by dihydrofolate reductase from lactobacillus casei

Two specific DNA binding sites for the enzyme dihydrofolate reductase from Lactobacillus casei have been located by means of an immunoprecipitation assay within a 2900-base pair L. casei DNA fragment containing the L. casei dihydrofolate reductase structural gene, which was previously cloned into pBR322. The inserted L. casei DNA was mapped using restriction endonucleases, and the location and orientation of the structural gene coding for L. casei dihydrofolate reductase were determined. The two specific binding sites map at the 5' end of the structural gene, approximately 100 base pairs upstream from the start of the coding region.     

Mutagenicity of chalks. A case in which the test results led to the improvement of the quality of commercial goods

The Adh gene from 4 formaldehyde-generated ADH-negative mutants of Drosophila melanogaster has been cloned and sequenced. All 4 mutants bear small deletions within the gene, ranging in size from 6 to 34 base pairs. 2 of the deletions lie within a 65-base pair intervening sequence and are accompanied by other aberrations. The other two are within the protein coding region of the gene. Some of these aberrations may be explained by a slipped mispairing mechanism.     

Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli.

In Escherichia coli, aroF, aroG, and aroH encode 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isozymes that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. In vitro chemical mutagenesis of the cloned aroG gene was used to identify residues and regions of the polypeptide essential for phenylalanine feedback inhibition.     

Repression of the aroF promoter by the TyrR repressor in Escherichia coli K-12: role of the ''upstream'' operator site

Three sequences are required for complete repression of the aroF promoter by the TyrR repressor protein. Two of these operator sites lie adjacent to each other and overlap the -35 region of the aroF promoter while the third lies about 70 base pairs upstream of the promoter. An aroF-cat (chloramphenicol acetyltransferase) gene fusion has been used to assay the effect of DNA insertions that alter the distance between the two promoter-proximal and the third, distal, operator sites on the repression of the aroF promoter in vivo. The distal site contributes to the repression of the promoter up to a distance of about 400 base pairs and its effect is not dependent on its separation from the first and second sites by an integral number of turns of the DNA helix.     

Escherichia coli promoters. I. Consensus as it relates to spacing class, specificity, repeat substructure, and three-dimensional organization

Fifty-two of the best characterized Escherichia coli promoters in the Hawley and McClure [1983) Nucleic Acids Res. 8, 2237-2255) listing were used to determine the distribution of information content in promoters and to describe the basic features underlying the existence of several different promoter spacing classes, which are defined by the number of bases separating the -35 and -10 regions. The contact regions at -35 and -10 do not, on the average, contain sufficient information to specify a promoter. The search for additional specifying bases led to two conclusions: 1) the consensus nucleotide sequence in the noncontact regions of a promoter appears to be distinct for each of the major promoter spacing classes; 2) promoters appear to contain a 15-20 base subset of the 40-50 additional optimal noncontact bases. This improved view of the extended consensus sequence allows the detection of a 10-base degenerate palindrome which may be the basic unit of promoter structure. Contiguous direct repeats of this sequence produce a sequence closely related to the consensus for the 18-base pair spacing class. This underlying structure is also evidenced in the 17- and 16-base pair spacing classes; however, the start points of the fourth and subsequent repetitions of the sequence element are moved one and two bases upstream, respectively, relative to their location in the 18-base pair spacing class. These consensus sequences, when viewed in a helical format, all present the opportunity for two alternative sets of a dyad repeat. The -35 region is common to both sets and is paired with an extended -10 region in one set and with a pseudo-10 region in the other. Possible implications of these arrangements are discussed.